Rui:LabNotes/Collaborations/2011-7-11
Jump to navigation
Jump to search
Prepare RNAseq library for sequencing
- Continue with lab note on 7/8/11
- Samples: PC1, PC2, GFP+, GFP-, E9.5, E11.5, E13.5m, E13.5f
- 75ul sheared DNA store at 0.5ml non-stick tube over the weekend
- Use NEB master mix kit for Library prep
End repair
Reaction sheared DNA 75ul Master mix buffer 10ul Enzyme mix 5ul H2O 10ul Total 100ul 20C for 30min
Purification with AMPure beads # 150ul beads to 100ul reaction (kapa lib prep kit suggested), mix 10 times, R.T for 8min # Place on magnetic stand for 5min # Remove sup., wash with 180ul fresh made 80% EtOH twice # Air-dry for 15min, re-suspend with 32ul Qiagen EB, mix 10 times, RT 2min, magnetic stand 2min # Transfer 30ul DNA from beads